Influence Of EPCs Mobilization And ERK/CREB Signaling Pathway On Myocardial Neovascularization In Rats With Acute Myocardial Infarction And The Interventional Effects Of Olmesartan Medoxomil

Objectives: In order to study the influence of EPCs mobilization and ERK/CREB signaling pathway on myocardial neovascularization in rats with acute myocardial infarction and the interventional effects of olmesartan medoxomil , the effects of olmesartan medoxomil on blood pressures, microvessle density (MVD) in myocardium, myocardial infarct area, vascular endothelium growth factor (VEGF) levels in peripherial blood, numbers of endothelial progenitor cells (EPCs) in peripherial blood and bone marrow and the expressions of myocardial p-ERK p-CREB proteins were explored.Methods:(1)Development of rat models with acute myocardial infarction and experimental grouping: Myocardial infarction (MI) was induced by ligation of the left coronary artery in male Sprague-Dawley rats. Twenty-four survivors within 24 hours after operation were randomized into three groups: The AMI group(n=8);The OML group(n=8), olmesartan medoxomil(1mg/kg/d)was administered;The OMH group(n=8), olmesartan medoxomil(3mg/kg/d)was administered. The Sham group was served as a control group(n=8). Olmesartan medoxomil was administered through gavage 24 hours after operation for 2 weeks. The Sham group and the AMI group were fed with vehicle in the same volume and the same method.(2)Contents surveyed:①Systolic blood pressures taken through tail artery:They were measured before operation, 24 hours after operation (before gavage) and week 1 and week 2 ;②Levels of serum VEGF: Blood samples were drawn through postorbital vein 2 weeks after operation . Levels of serum VEGF were measured using enzyme linked-immunoabsorbent assay ( ELISA) ;③Numbers of EPCs in peripherial blood and bone marrow:Peripherial blood was drawn through postorbital vein 1 week after operation. Blood was drawn via heart 2 weeks after operation. Bone marrow was harvested at 2 weeks after operation. Cells were separated, cultured, dyed and identified with both 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindo-carbocyanine perchlorate labeled acetylated Low Density Lipoprotein ( DiI-acLDL)and fluorescein isothiocyanate -conjugated Bandeiraea simplicifolia-1 lectin(FITC-BS-1);④Pathological changes of heart tissue and myocardial infarct area: Left ventricles were cut along cross-sections immediately after the death of rats and then made into paraffin sections. The percentage between the lengths of cardiac scar and those of circumference stands for the myocardial infarct area by applying HE staining;⑤Numbers of MVD: Numbers of MVD were calculated by immunohistochemical method using antibody of factorⅧrelated antigen;⑥Expressions of myocardial p-ERK and p-CREB proteins:They were examined by immunohistochemical method.Results: (1) Systolic blood pressures taken through tail artery:Systolic blood pressures taken through tail artery at different time did not have significant differences in different group; (2) Levels of serum VEGF: Compared with those in the Sham group, levels of serum VEGF in the AMI group, the OML group and the OMH group were much higher . The differences were significant;Compared with the AMI group, the OML group and the OMH group had lower levels of VEGF, but the differences were insignificant;(3) Numbers of EPCs in peripherial blood: There were no significant differences between numbers of EPCs in the Sham group at week 1 and week 2;The same with the AMI group. In the OML group and the OMH group numbers of circulating EPCs at week 2 were larger than those at week 1 and their differences were significant; Numbers of circulating EPCs in the AMI group at the same time points were significantly larger than those in Group Sham; Numbers of circulating EPCs at the same time points in the OML group and the OMH group were much larger compared with those in the AMI group; Furthermore, olmesartan medoxomil can increse the number of circulating EPCs in a dose-dependant manner and the differences between the OML group and the OMH group were significant; (4) Numbers of EPCs in bone marrow:Numbers of EPCs in bone marrow in the AMI group at week 2 were much larger than those in the Sham group;The OML group had larger numbers of EPCs in bone marrow than the AMI group;the same with the OMH group. Olmesartan medoxomil can increase the numbers of EPCs in bone marrow in a dose-dependant manner;(5) HE dyeing of heart tissue and the calculation of myocardial infarction area:Heart tissue showed normal HE dyeing features in Group Sham;On the other hand, at infarction zones in the AMI group, the OML group and the OMH group myocardial dissolution, fragmentation, inflammatory infiltration, fibroblast infiltration and formation of granalation tissue were obviously seen. At non-infarction zones cardiac muscles arranged regularly;Olmesartan medoxomil can decrease the size of myocardial infarct in a dose-dependant manner; (6) MVD: MVD at week 2 was significantly higher in the AMI group than that in the Sham group. Olmesartan medoxomil can result in a substantial increase in MVD in a dose-dependant manner; (7) The expressions of myocardial p-ERK and p-CREB proteins: The results of immunohistochemical staining assay showed that the expressions of myocardial p-ERK and p-CREB proteins were higher in the AMI group than in the Sham group. Olmesartan medoxomil can decrease the expressions of myocardial p-ERK and p-CREB proteins in a dose-dependant manner. Conclusions: (1) The increase of myocardial neovascularization after AMI is a"physiologically"protective reaction which can not stop the progression of AMI;(2) The increase of myocardial neovascularization after AMI is associated with elevated serum VEGF and mobilization of EPCs in bone marrow;(3) The increased expressions of myocardial p-ERK and p-CREB proteins after AMI shows the activation of ERK/CREB signaling pathway after AMI and it may promote myocardial neovascularization;(4) Olmesartan medoxomil can enhance myocardial neovascularization and decrease the size of myocardial infarction. The possibly underlying mechanism may be that Olmesartan medoxomil can enhance the mobilization of EPCs in bone marrow; (5) Olmesartan medoxomil can decrease the expressions of myocardial p-ERK and p-CREB proteins in AMI rats. Olmesartan medoxomil may inhibit the activation of ERK/CREB signaling pathway through the inhibition of AT1R. The association between the inhibition of ERK/CREB singaling pathway and myocardial neovascularization needs to be further investigated; (6) The effect of olmesartan medoxomil on myocardial neovascularization in AMI rats is independent on blood pressure lowering and VEGF.